Listeriolysin O Is Strongly Immunogenic Independently of Its Cytotoxic Activity

The presentation of microbial protein antigens by Major Histocompatibility Complex (MHC) molecules is essential for the development of acquired immunity to infections. However, most biochemical studies of antigen processing and presentation deal with a few relatively inert non-microbial model antigens. The bacterial pore-forming toxin listeriolysin O (LLO) is paradoxical in that it is cytotoxic at nanomolar concentrations as well as being the source of dominant CD4 and CD8 T cell epitopes following infection with Listeria monocytogenes. Here, we examined the relationship of LLO toxicity to its antigenicity and immunogenicity. LLO offered to antigen presenting cells (APC) as a soluble protein, was presented to CD4 T cells at picomolar to femtomolar concentrations- doses 3000–7000-fold lower than free peptide. This presentation required a dose of LLO below the cytotoxic level. Mutations of two key tryptophan residues reduced LLO toxicity by 10–100-fold but had no effect on its presentation to CD4 T cells. Thus there was a clear dissociation between the cytotoxic properties of LLO and its very high antigenicity. Presentation of LLO to CD8 T cells was not as robust as that seen in CD4 T cells, but still occurred in the nanomolar range. APC rapidly bound and internalized LLO, then disrupted endosomal compartments within 4 hours of treatment, allowing endosomal contents to access the cytosol. LLO was also immunogenic after in vivo administration into mice. Our results demonstrate the strength of LLO as an immunogen to both CD4 and CD8 T cells

Reduced Secretion of YopJ by Yersinia Limits In Vivo Cell Death but Enhances Bacterial Virulence

Numerous microbial pathogens modulate or interfere with cell death pathways in cultured cells. However, the precise role of host cell death during in vivo infection remains poorly understood. Macrophages infected by pathogenic species of Yersinia typically undergo an apoptotic cell death. This is due to the activity of a Type III secreted effector protein, designated YopJ in Y. pseudotuberculosis and Y. pestis, and YopP in the closely related Y. enterocolitica. It has recently been reported that Y. enterocolitica YopP shows intrinsically greater capacity for being secreted than Y. pestis YopJ, and that this correlates with enhanced cytotoxicity observed for high virulence serotypes of Y. enterocolitica. The enzymatic activity and secretory capacity of YopP from different Y. enterocolitica serotypes have been shown to be variable. However, the underlying basis for differential secretion of YopJ/YopP, and whether reduced secretion of YopJ by Y. pestis plays a role in pathogenesis during in vivo infection, is not currently known. It has also been reported that similar to macrophages, Y. enterocolitica infection of dendritic cells leads to YopP-dependent cell death. We demonstrate here that in contrast to Y. enterocolitica, Y. pseudotuberculosis infection of bone marrow–derived dendritic cells does not lead to increased cell death. However, death of Y. pseudotuberculosis–infected dendritic cells is enhanced by ectopic expression of YopP in place of YopJ. We further show that polymorphisms at the N-terminus of the YopP/YopJ proteins are responsible for their differential secretion, translocation, and consequent cytotoxicity. Mutation of two amino acids in YopJ markedly enhanced both translocation and cytotoxicity. Surprisingly, expression of YopP or a hypersecreted mutant of YopJ in Y. pseudotuberculosis resulted in its attenuation in oral mouse infection. Complete absence of YopJ also resulted in attenuation of virulence, in accordance with previous observations. These findings suggest that control of cytotoxicity is an important virulence property for Y. pseudotuberculosis, and that intermediate levels of YopJ-mediated cytotoxicity are necessary for maximal systemic virulence of this bacterial pathogen

Anthrax Lethal Toxin Disrupts Intestinal Barrier Function and Causes Systemic Infections with Enteric Bacteria

A variety of intestinal pathogens have virulence factors that target mitogen activated protein kinase (MAPK) signaling pathways, including Bacillus anthracis. Anthrax lethal toxin (LT) has specific proteolytic activity against the upstream regulators of MAPKs, the MAPK kinases (MKKs). Using a murine model of intoxication, we show that LT causes the dose-dependent disruption of intestinal epithelial integrity, characterized by mucosal erosion, ulceration, and bleeding. This pathology correlates with an LT-dependent blockade of intestinal crypt cell proliferation, accompanied by marked apoptosis in the villus tips. C57BL/6J mice treated with intravenous LT nearly uniformly develop systemic infections with commensal enteric organisms within 72 hours of administration. LT-dependent intestinal pathology depends upon its proteolytic activity and is partially attenuated by co-administration of broad spectrum antibiotics, indicating that it is both a cause and an effect of infection. These findings indicate that targeting of MAPK signaling pathways by anthrax LT compromises the structural integrity of the mucosal layer, serving to undermine the effectiveness of the intestinal barrier. Combined with the well-described immunosuppressive effects of LT, this disruption of the intestinal barrier provides a potential mechanism for host invasion via the enteric route, a common portal of entry during the natural infection cycle of Bacillus anthracis

MyD88 and STING Signaling Pathways Are Required for IRF3-Mediated IFN-β Induction in Response to Brucella abortus Infection

Type I interferons (IFNs) are cytokines that orchestrate diverse immune responses to viral and bacterial infections. Although typically considered to be most important molecules in response to viruses, type I IFNs are also induced by most, if not all, bacterial pathogens. In this study, we addressed the role of type I IFN signaling during Brucella abortus infection, a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. Herein, we have shown that B. abortus induced IFN-β in macrophages and splenocytes. Further, IFN-β induction by Brucella was mediated by IRF3 signaling pathway and activates IFN-stimulated genes via STAT1 phosphorylation. In addition, IFN-β expression induced by Brucella is independent of TLRs and TRIF signaling but MyD88-dependent, a pathway not yet described for Gram-negative bacteria. Furthermore, we have identified Brucella DNA as the major bacterial component to induce IFN-β and our study revealed that this molecule operates through a mechanism dependent on RNA polymerase III to be sensed probably by an unknown receptor via the adaptor molecule STING. Finally, we have demonstrated that IFN-αβR KO mice are more resistant to infection suggesting that type I IFN signaling is detrimental to host control of Brucella. This resistance phenotype is accompanied by increased IFN-γ and NO production by IFN-αβR KO spleen cells and reduced apoptosis

Bacillus anthracis Spore Surface Protein BclA Mediates Complement Factor H Binding to Spores and Promotes Spore Persistence

Spores of Bacillus anthracis, the causative agent of anthrax, are known to persist in the host lungs for prolonged periods of time, however the underlying mechanism is poorly understood. In this study, we demonstrated that BclA, a major surface protein of B. anthracis spores, mediated direct binding of complement factor H (CFH) to spores. The surface bound CFH retained its regulatory cofactor activity resulting in C3 degradation and inhibition of downstream complement activation. By comparing results from wild type C57BL/6 mice and complement deficient mice, we further showed that BclA significantly contributed to spore persistence in the mouse lungs and dampened antibody responses to spores in a complement C3-dependent manner. In addition, prior exposure to BclA deletion spores (ΔbclA) provided significant protection against lethal challenges by B. anthracis, whereas the isogenic parent spores did not, indicating that BclA may also impair protective immunity. These results describe for the first time an immune inhibition mechanism of B. anthracis mediated by BclA and CFH that promotes spore persistence in vivo. The findings also suggested an important role of complement in persistent infections and thus have broad implications

Burkholderia cenocepacia Type VI Secretion System Mediates Escape of Type II Secreted Proteins into the Cytoplasm of Infected Macrophages

<div><p><em>Burkholderia cenocepacia</em> is an opportunistic pathogen that survives intracellularly in macrophages and causes serious respiratory infections in patients with cystic fibrosis. We have previously shown that bacterial survival occurs in bacteria-containing membrane vacuoles (BcCVs) resembling arrested autophagosomes. Intracellular bacteria stimulate IL-1β secretion in a caspase-1-dependent manner and induce dramatic changes to the actin cytoskeleton and the assembly of the NADPH oxidase complex onto the BcCV membrane. A Type 6 secretion system (T6SS) is required for these phenotypes but surprisingly it is not required for the maturation arrest of the BcCV. Here, we show that macrophages infected with <em>B. cenocepacia</em> employ the NLRP3 inflammasome to induce IL-1β secretion and pyroptosis. Moreover, IL-1β secretion by <em>B. cenocepacia-</em>infected macrophages is suppressed in deletion mutants unable to produce functional Type VI, Type IV, and Type 2 secretion systems (SS). We provide evidence that the T6SS mediates the disruption of the BcCV membrane, which allows the escape of proteins secreted by the T2SS into the macrophage cytoplasm. This was demonstrated by the activity of fusion derivatives of the T2SS-secreted metalloproteases ZmpA and ZmpB with adenylcyclase. Supporting this notion, ZmpA and ZmpB are required for efficient IL-1β secretion in a T6SS dependent manner. ZmpA and ZmpB are also required for the maturation arrest of the BcCVs and bacterial intra-macrophage survival in a T6SS-independent fashion. Our results uncover a novel mechanism for inflammasome activation that involves cooperation between two bacterial secretory pathways, and an unanticipated role for T2SS-secreted proteins in intracellular bacterial survival.</p> </div